Macrophage-derived mir-100-5p orchestrates synovial proliferation and inflammation in rheumatoid arthritis through mTOR signaling.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial inflammation, causing substantial disability and reducing life quality. While macrophages are widely appreciated as a master regulator in the inflammatory response of RA, the precise mechanisms underlying the regulation of proliferation and inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS) remain elusive. Here, we provide extensive evidence to demonstrate that macrophage contributes to RA microenvironment remodeling by extracellular vesicles (sEVs) and downstream miR-100-5p/ mammalian target of rapamycin (mTOR) axis. RESULTS: We showed that bone marrow derived macrophage (BMDM) derived-sEVs (BMDM-sEVs) from collagen-induced arthritis (CIA) mice (cBMDM-sEVs) exhibited a notable increase in abundance compared with BMDM-sEVs from normal mice (nBMDM-sEVs). cBMDM-sEVs induced significant RA-FLS proliferation and potent inflammatory responses. Mechanistically, decreased levels of miR-100-5p were detected in cBMDM-sEVs compared with nBMDM-sEVs. miR-100-5p overexpression ameliorated RA-FLS proliferation and inflammation by targeting the mTOR pathway. Partial attenuation of the inflammatory effects induced by cBMDM-sEVs on RA-FLS was achieved through the introduction of an overexpression of miR-100-5p. CONCLUSIONS: Our work reveals the critical role of macrophages in exacerbating RA by facilitating the transfer of miR-100-5p-deficient sEVs to RA-FLS, and sheds light on novel disease mechanisms and provides potential therapeutic targets for RA interventions.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation, posing challenges in the development of effective treatments. Nuciferine, an alkaloid found in lotus leaf, has shown promising anti-inflammatory and anti-tumor effects, yet its efficacy in RA treatment remains unexplored. This study investigated the antiproliferative effects of nuciferine on the MH7A cell line, a human RA-derived fibroblast-like synoviocyte, revealing its ability to inhibit cell proliferation, promote apoptosis, induce apoptosis, and cause G1/S phase arrest. Additionally, nuciferine significantly reduced the migration and invasion capabilities of MH7A cells. The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis (CIA) rat model, where it markedly alleviated joint swelling, synovial hyperplasia, cartilage injury, and inflammatory infiltration. Nuciferine also improved collagen-induced bone erosion, decreased pro-inflammatory cytokines and serum immunoglobulins (IgG, IgG1, IgG2a), and restored the balance between T helper (Th) 17 and regulatory T cells in the spleen of CIA rats. These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Deconvolution of synovial myeloid cell subsets across pathotypes and role of COL3A1+ macrophages in rheumatoid arthritis remission.

Background: Monocyte/macrophage (Mo/Mp) is a critical cell population involved in immune modulation of rheumatoid synovitis (RA) across different pathotypes. This study aims to investigate the contribution of Mo/Mp clusters to RA activity, and the biological function of particular subtypes in RA remission. Methods: We integrated single-cell RNA sequencing datasets from 4 published and 1 in-house studies using Liger selected by comparison. We estimated the abundance of Mo/Mp subtypes in bulk RNA-seq data from the 81 patients of the Pathobiology of Early Arthritis Cohort (PEAC) using deconvolution analysis. Correlations between Mo/Mp subtypes and RA clinical metrics were assessed. A particular cell type was identified using multicolor immunofluorescence and flow cytometry in vivo and successfully induced from a cell line in vitro. Potential immune modulation function of it was performed using immunohistochemical staining, adhesion assay, and RT-qPCR. Results: We identified 8 Mo/Mp clusters. As a particular subtype among them, COL3A1+ Mp (CD68+, COL3A1+, ACTA2-) enriched in myeloid pathotype and negatively correlated with RA severity metrics in all pathotypes. Flow cytometry and multicolor immunofluorescence evidenced the enrichment and M2-like phenotype of COL3A1+ Mp in the myeloid pathotype. Further assays suggested that COL3A1+ Mp potentially attenuates RA severity via expressing anti-inflammatory cytokines, enhancing Mp adhesion, and forming a physical barrier at the synovial lining. Conclusion: This study reported unexplored associations between different pathologies and myeloid cell subtypes. We also identified a fibroblast-and-M2-like cluster named COL3A1+ Mp, which potentially contributes to synovial immune homeostasis. Targeting the development of COL3A1+ Mp may hold promise for inducing RA remission.

Tetramethylpyrazine alleviates hypoxia-induced proliferation, migration, and inflammatory response of fibroblast-like synoviocytes via inhibiting the HIF-1α- circCDC42BPB pathway.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which might trigger cartilage, bone damage, and disability. Recent studies have suggested that Tetramethylpyrazine (TMP), an alkaloid monomer isolated from the rhizome of the traditional herbal medicine Ligusticum wallichii Franch, exerts a broad spectrum of pharmacological properties, containing anti-inflammatory. This study aimed to analyze the role and underlying mechanism of TMP in RA. METHODS: Under Hypoxia condition, RA-Fibroblast-like synoviocyte (FLS) were treated with TMP at different doses. Cell viability, proliferation, cell cycle progression, and migration were detected using Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry assay, wound healing assay, and transwell assay. Cyclin D1, Proliferating cell nuclear antigen (PCNA), Matrix metalloproteinase-2 (MMP2), MMP9, and hypoxia-inducible factor-1α (HIF-1α) protein levels were measured using western blot assay. Interleukin-6 (IL-6) and IL-8 were evaluated using ELISA. Circular RNA (circRNA) hsa_circ_0005178 (circCDC42BPB), CDC42BPB, and HIF-1α expression were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Binding between HIF-1α and CDC42BPB promoter was predicted by JASPAR and verified using dual-luciferase reporter and Chromatin immunoprecipitation (ChIP) assays. RESULTS: TMP might hinder FLS proliferation, cycle progression, migration, and inflammatory response under hypoxic conditions. CircCDC42BPB expression was increased in RA patients and RA-FLSs treated with hypoxia, while its level was obviously reduced in RA-FLSs treated with hypoxia and TMP. TMP might abolish hypoxia-induced circCDC42BPB expression. Upregulation of circCDC42BPB might partially overturn the repression of TMP on hypoxia-caused RA-FLS damage. TMP might regulate circCDC42BPB level via HIF-1α in RA-FLSs under hypoxic conditions. CONCLUSION: TMP might block RA-FLS injury partly via regulating the HIF-1α- circCDC42BPB pathway, providing a promising therapeutic target for RA.

HIGHLIGHTS: • TMP suppressed hypoxia-induced RA-FLS growth and inflammatory response.• TMP might repress circCDC42BPB expression in RA-FLSs under hypoxic conditions.• TMP might inhibit HIF-1α-induced circCDC42BPB transcription under hypoxic conditions.

pH-responsive size-adjustable liposomes induce apoptosis of fibroblasts and macrophages for rheumatoid arthritis treatment.

In rheumatoid arthritis (RA), macrophages infiltrate joints, while fibroblast-like synovial cells proliferate abnormally, forming a barrier against drug delivery, which hinders effective drug delivery to joint focus. Here we firstly designed a pH-responsive size-adjustable nanoparticle, composed by methotrexate (MTX)-human serum albumin (HSA) complex coating with pH-responsive liposome (Lipo/MTX-HSA) for delivering drugs specifically to inflamed joints in acidic environments. We showed in vitro that the nanoparticles can induce mitochondrial dysfunction, promote apoptosis of fibroblast-like synoviocytes and macrophages, further reduce the secretion of inflammatory factors (TNF-α, IL-1ß, MMP-9), and regulate the inflammatory microenvironment. We also demonstrated similar effects in a rat model of arthritis, in which Lipo/MTX-HSA accumulated in arthritic joints, and at low pH, liposome phospholipid bilayer cleavage released small-sized MTX-HSA, which effectively reduced the number of fibroblast-synoviocytes and macrophages in joints, alleviated joint inflammation, and repaired bone erosion. These findings suggest that microenvironment-responsive size-adjustable nanoparticles show promise as a treatment against rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Abnormal proliferation of fibroblast synoviocytes poses a physical barrier to effective nanoparticle delivery. We designed size-adjustable nano-delivery systems by preparing liposomes with cholesterol hemisuccinate (CHEM), which were subsequently loaded with small-sized albumin nanoparticles encapsulating the cytotoxic drug MTX (MTX-HSA), termed Lipo/MTX-HSA. Upon tail vein injection, Lipo/MTX-HSA could be aggregated at the site of inflammation via the ELVIS effect in the inflamed joint microenvironment. Specifically, intracellular acidic pH-triggered dissociation of liposomes promoted the release of MTX-HSA, which was further targeted to fibroblasts or across fibroblasts to macrophages to exert anti-inflammatory effects. The results showed that liposomes with adjustable particle size achieved efficient drug delivery, penetration and retention in joint sites; the strategy exerted significant anti-inflammatory effects in the treatment of rheumatoid arthritis by inducing mitochondrial dysfunction to promote apoptosis in fibrosynoviocytes and macrophages.

METTL3 knockdown suppresses RA-FLS activation through m6A-YTHDC2-mediated regulation of AMIGO2.

The dysregulation of N6-methyladenosine (m6A) on mRNAs is involved in the pathogenesis of rheumatoid arthritis (RA). Methyltransferase-like 3 (METTL3), serving as a central m6A methyltransferase, is highly expressed in macrophages, synovial tissues and RA fibroblast-like synoviocytes (RA-FLS) of RA patients. However, METTL3-mediated m6A modification on target mRNAs and the molecular mechanisms involved in RA-FLS remain poorly defined. Our research demonstrated that METTL3 knockdown decreased the proliferation, migratory and invasive abilities of RA-FLS. Notably, we identified the adhesion molecule with Ig like domain 2 (AMIGO2) as a probable downstream target of both METTL3 and YTH Domain Containing 2 (YTHDC2) in RA-FLS. We revealed that AMIGO2 augmented the activation of RA-FLS and can potentially reverse the phenotypic effects induced by the knockdown of either METTL3 or YTHDC2. Mechanistically, METTL3 knockdown decreased m6A modification in the 5'-untranslated region (5'UTR) of AMIGO2 mRNA, which diminished its interaction with YTHDC2 in RA-FLS. Our findings unveiled that silencing of METTL3 inhibited the proliferation and aggressive behaviors of RA-FLS by downregulating AMIGO2 expression in an m6A-YTHDC2 dependent mechanism, thereby underscoring the pivotal role of the METTL3-m6A-YTHDC2-AMIGO2 axis in modulating RA-FLS phenotypes.

Lovastatin impairs cellular proliferation and enhances hyaluronic acid production in fibroblast-like synoviocytes.

INTRODUCTION: Statins have demonstrated chondroprotective effects by reducing inflammation and mitigating extracellular matrix degradation. However, statins are also reported to be cytotoxic to several types of cells. Early-onset osteoarthritis (OA) is characterized by synovial inflammation, which adversely affects hyaluronan (HA) production in fibroblast-like synoviocytes (FLSs). Nevertheless, the precise effects of statins on the synovium remain unclear. METHODS: This study investigated the impact of lovastatin on human FLSs, and HA secretion-related genes, signaling pathways, and production were evaluated. RESULTS: The findings revealed that high doses of lovastatin (20 or 40 µM) decreased FLS viability and increased cell death. FLS proliferation ceased when cultured in a medium containing 5 or 10 µM lovastatin. mRNA expression analysis demonstrated that lovastatin (5 and 10 µM) upregulated the gene level of hyaluronan synthase 1 (HAS1), HAS2, and proteoglycan 4 (PRG4), but not HAS3. While the expression of multidrug resistance-associated protein 5 transporter gene remained unaffected, both inward-rectifying potassium channel and acid-sensing ion channel 3 were upregulated. Western blot further confirmed that lovastatin increased the production of HAS1 and PRG4, and activated the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Additionally, lovastatin elevated intracellular cAMP levels and HA production in FLSs. CONCLUSION: Lovastatin impairs cellular proliferation but enhances HA production in human FLSs.

Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

Asiatic acid inhibits rheumatoid arthritis fibroblast-like synoviocyte growth through the Nrf2/HO-1/NF-κB signaling pathway.

Asiatic acid (AA) is generally recognized in the treatment of various diseases and has significant advantages in the treatment of various inflammatory diseases. The treatment of rheumatoid arthritis (RA) with AA is a completely new entry point. RA is a complex autoimmune inflammatory disease, and despite the involvement of different immune and nonimmune cells in the pathogenesis of RA, fibroblast-like synoviocytes (FLS) play a crucial role in the progression of the disease. si-Nrf2 was transfected in RA-FLS and the cells were treated with AA. MTT assay and colony formation assay were used to detect the effect of AA on the viability and formation of clones of RA-FLS, respectively. Moreover, the apoptosis of RA-FLS was observed by Hoechst 33342 staining and flow cytometry. Western blot was applied to measure the expression of the Nrf2/HO-1/NF-κB signaling pathway-related proteins. Compared with the control group, RA-FLS proliferation, and clone formation were significantly inhibited by the increase of AA concentration, and further experiments showed that AA-induced apoptosis of RA-FLS. In addition, AA activated the Nrf2/HO-1 pathway to inhibit NF-κB protein expression. However, the knockdown of Nrf2 significantly offsets the effects of AA on the proliferation, apoptosis, and Nrf2/HO-1/NF-κB signaling pathway of RA-FLS cells. AA can treat RA by inhibiting the proliferation and inducing the apoptosis of RA-FLS. The mechanism may be related to the activation of the Nrf2/HO-1/NF-κB pathway.

Revolutionizing osteoarthritis treatment: How mesenchymal stem cells hold the key.

Osteoarthritis (OA) is a multifaceted disease characterized by imbalances in extracellular matrix metabolism, chondrocyte and synoviocyte senescence, as well as inflammatory responses mediated by macrophages. Although there have been notable advancements in pharmacological and surgical interventions, achieving complete remission of OA remains a formidable challenge, oftentimes accompanied by significant side effects. Mesenchymal stem cells (MSCs) have emerged as a promising avenue for OA treatment, given their ability to differentiate into chondrocytes and facilitate cartilage repair, thereby mitigating the impact of an inflammatory microenvironment induced by macrophages. This comprehensive review aims to provide a concise overview of the diverse roles played by MSCs in the treatment of OA, while elucidating the underlying mechanisms behind these contributions. Specifically, the roles include: (a) Promotion of chondrocyte and synoviocyte regeneration; (b) Inhibition of extracellular matrix degradation; (c) Attenuating the macrophage-induced inflammatory microenvironment; (d) Alleviation of pain. Understanding the multifaceted roles played by MSCs in OA treatment is paramount for developing novel therapeutic strategies. By harnessing the regenerative potential and immunomodulatory properties of MSCs, it may be possible to devise more effective and safer approaches for managing OA. Further research and clinical studies are warranted to optimize the utilization of MSCs and realize their full potential in the field of OA therapeutics.

Metabolic changes in fibroblast-like synoviocytes in rheumatoid arthritis: state of the art review.

Fibroblast-like synoviocytes (FLS) are important components of the synovial membrane. They can contribute to joint damage through crosstalk with inflammatory cells and direct actions on tissue damage pathways in rheumatoid arthritis (RA). Recent evidence suggests that, compared with FLS in normal synovial tissue, FLS in RA synovial tissue exhibits significant differences in metabolism. Recent metabolomic studies have demonstrated that metabolic changes, including those in glucose, lipid, and amino acid metabolism, exist before synovitis onset. These changes may be a result of increased biosynthesis and energy requirements during the early phases of the disease. Activated T cells and some cytokines contribute to the conversion of FLS into cells with metabolic abnormalities and pro-inflammatory phenotypes. This conversion may be one of the potential mechanisms behind altered FLS metabolism. Targeting metabolism can inhibit FLS proliferation, providing relief to patients with RA. In this review, we aimed to summarize the evidence of metabolic changes in FLS in RA, analyze the mechanisms of these metabolic alterations, and assess their effect on RA phenotype. Finally, we aimed to summarize the advances and challenges faced in targeting FLS metabolism as a promising therapeutic strategy for RA in the future.

The FTO-CMPK2 Pathway in Fibroblast-like Synoviocytes Modulates Rheumatoid Arthritis Synovial Inflammation and Cartilage Homeostasis via mtDNA Regulation.

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.

Inflammatory Fibroblast-Like Synoviocyte-Derived Exosomes Aggravate Osteoarthritis via Enhancing Macrophage Glycolysis.

The severity of osteoarthritis (OA) and cartilage degeneration is highly associated with synovial inflammation. Although recent investigations have revealed a dysregulated crosstalk between fibroblast-like synoviocytes (FLSs) and macrophages in the pathogenesis of synovitis, limited knowledge is available regarding the involvement of exosomes. Here, increased exosome secretion is observed in FLSs from OA patients. Notably, internalization of inflammatory FLS-derived exosomes (inf-exo) can enhance the M1 polarization of macrophages, which further induces an OA-like phenotype in co-cultured chondrocytes. Intra-articular injection of inf-exo induces synovitis and exacerbates OA progression in murine models. In addition, it is demonstrated that inf-exo stimulation triggers the activation of glycolysis. Inhibition of glycolysis using 2-DG successfully attenuates excessive M1 polarization triggered by inf-exo. Mechanistically, HIF1A is identified as the determinant transcription factor, inhibition of which, both pharmacologically or genetically, relieves macrophage inflammation triggered by inf-exo-induced hyperglycolysis. Furthermore, in vivo administration of an HIF1A inhibitor alleviates experimental OA. The results provide novel insights into the involvement of FLS-derived exosomes in OA pathogenesis, suggesting that inf-exo-induced macrophage dysfunction represents an attractive target for OA therapy.

LncRNA HOTTIP regulates TLR4 promoter methylation by recruiting H3K4 methyltransferase MLL1 to affect apoptosis and inflammatory response of fibroblast-like synoviocyte in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and the role of HOXA transcript at the distal tip (HOTTIP) in its pathogenesis remains underexplored. This study investigates the mechanism by which HOTTIP influences apoptosis and the inflammatory response of fibroblast-like synoviocytes (FLS). An RA mouse model was established, and clinical scores were analyzed. Pathological changes in synovial tissues, bone mineral density (BMD) of the paws, serum tartrate-resistant acid phosphatase (TRAP) activity, and TNF-α and IL-1ß levels were assessed. FLS were transfected, and cell proliferation and apoptosis were examined. The RNA-pull-down assay determined HOTTIP's interaction with mixed-lineage leukemia 1 (MLL1), while RNA immunoprecipitation assay measured HOTTIP expression pulled down by MLL1. The levels of MLL1 and toll-like receptor 4 (TLR4) after MLL1 overexpression based on HOTTIP silencing were determined. Chromatin immunoprecipitation (ChIP) was performed with H3K4me3 as an antibody, followed by the evaluation of TLR4 expression. HOTTIP expression was elevated in RA mouse synovial tissues. Inhibition of HOTTIP led to reduced clinical scores, inflammatory infiltration, synovial hyperplasia, TRAP activity, and TNF-α and IL-1ß levels, along with increased BMD. In vitro Interference with HOTTIP suppressed RA-FLS apoptosis and inflammation. HOTTIP upregulated TLR4 expression by recruiting MLL1 to facilitate TLR4 promoter methylation. MLL1 overexpression reversed HOTTIP silencing-mediated repression of RA-FLS apoptosis. Activation of H3K4 methylation counteracted HOTTIP knockout, ameliorating the inflammatory response. HOTTIP regulates TLR4 expression by recruiting MLL1, leading to TLR4 promoter methylation, thereby suppressing RA-FLS proliferation and inducing cell apoptosis and inflammatory response in RA.

LIGHT regulated gene expression in rheumatoid synovial fibroblasts.

BACKGROUND: Synovial hyperplasia caused by rheumatoid arthritis (RA), an autoimmune inflammatory disease, leads to the destruction of the articular cartilage and bone. A member of the tumor necrosis factor superfamily, Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (LIGHT) has been shown to correlate with the pathogenesis of RA. METHODS: We used cDNA microarray analysis to compare the expression of genes in rheumatoid fibroblast-like synoviocytes with and without LIGHT stimulation. RESULTS: Significant changes in gene expression (P-values < 0.05 and fold change ≥ 2.0) were associated mainly with biological function categories of glycoprotein, glycosylation site as N-linked, plasma membrane part, integral to plasma membrane, intrinsic to plasma membrane, signal, plasma membrane, signal peptide, alternative splicing, and topological domain as extracellular. CONCLUSIONS: Our results indicate that LIGHT may regulate the expression in RA-FLS of genes which are important in the differentiation of several cell types and in cellular functions.

Fibroblast-like synoviocytes-derived exosomal circFTO deteriorates rheumatoid arthritis by enhancing N6-methyladenosine modification of SOX9 in chondrocytes.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes disability worldwide. Exosomes released by fibroblast-like synoviocytes in RA (RA-FLSs-Exos) play a role in the development of RA, and circular RNAs (circRNAs) are important for RA progression. This study aimed to investigate the molecular mechanisms underlying the effects of RA-FLSs-Exos in RA and identify the potential pathway responsible for these effects. METHODS: We initially conducted microarray analysis to identify dysregulated circRNAs in exosomes associated with RA. We then co-cultured isolated RA-FLSs-Exos with chondrocytes to examine their role in RA. In vivo experiments were performed using collagen-induced arthritis mouse models, and circFTO knockdown was achieved through intra-articular injection of AAV5 vectors. RESULTS: Our findings revealed increased expression of circFTO in both RA-FLSs-Exos and synovial tissues from patients with RA. Exosomal circFTO hindered chondrocyte proliferation, migration, and anabolism while promoting apoptosis and catabolism. Mechanistically, we discovered that circFTO facilitates the formation of methyltransferases complex to suppress SRY-related high-mobility group box 9 (SOX9) expression with assistance from YTH domain family 2 (YTHDF2) through an m6A-dependent mechanism. Furthermore, inhibition of circFTO improved symptoms of RA in vivo. CONCLUSION: Taken together, our study demonstrates that exosomal circFTO derived from FLSs contributes to the progression of RA by targeting SOX9. These findings highlight a promising target for treating RA.

Amelioration of inflammation through reduction of oxidative stress in rheumatoid arthritis by treating fibroblast-like synoviocytes (FLS) with DMF-loaded PLGA nanoparticles.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory condition, and Dimethyl fumarate (DMF) is known for inducing antioxidant enzymes and reducing reactive oxygen species (ROS). Fibroblast-like synoviocytes (FLS) contribute to joint damage by releasing interleukins (IL-1ß, IL-6, and IL-8) in response to ROS. Given ROS's impact on FLS acquiring an invasive phenotype, our study explored the effects of poly lactic-co-glycolic acid (PLGA) nanoparticles containing DMF on the expression of the HO-1 enzyme and the inflammatory cytokines IL-1ß, IL-6, and IL-8 in FLS cells. METHODS: In this study, we evaluated and compared the impact of Free-DMF and PLGA-DMF, on the gene expression of the HO-1 and inflammatory cytokines (IL-1ß, IL-6, and IL-8) in FLS cells derived from 13 patients with rheumatoid arthritis. qRT-PCR method was used to quantify the gene expression levels. RESULTS: PLGA-DMF nanoparticles demonstrated a significant increase in HO-1 expression and a significant decrease in IL-1ß gene expression. Also, a significant decrease in IL-6 gene expression was seen under the effect of Free-DMF. These results indicate the potential effectiveness of PLGA-DMF nanoparticles in reducing inflammation and improving rheumatoid arthritis symptoms. DISCUSSION: According to the findings, PLGA-DMF nanoparticles are expected to be effective in reducing inflammation and improving the symptoms of rheumatoid arthritis. Also, further studies on other factors affected by oxidative stress such as cell invasion factors and survival factors after the effect of PLGA-DMF nanoparticle are recommended.

EPO promotes the progression of rheumatoid arthritis by inducing desialylation via increasing the expression of neuraminidase 3.

OBJECTIVE: Erythropoietin (EPO) known as an erythrocyte-stimulating factor is increased in patients with rheumatoid arthritis (RA). Nevertheless, the function of EPO in the process of RA and relative mechanism needs to be further clarified. METHODS: The level of EPO in serum and synovial fluid from patients with RA and healthy controls was determined by . Collagen-induced arthritis (CIA) mice were constructed to confirm the role of EPO on RA pathogenesis. Differentially expressed genes (DEGs) of EPO-treated fibroblast-like synoviocyte (FLS) were screened by transcriptome sequencing. The transcription factor of neuraminidase 3 (NEU3) of DEGs was verified by double luciferase reporting experiment, DNA pulldown, electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR (qPCR) assay. RESULTS: The overexpression of EPO was confirmed in patients with RA, which was positively associated with Disease Activity Score 28-joint count. Additionally, EPO intervention could significantly aggravate the joint destruction in CIA models. The upregulation of NEU3 was screened and verified by transcriptome sequencing and qPCR in EPO-treated FLS, and signal transducer and activator of transcription 5 was screened and verified to be the specific transcription factor of NEU3. EPO upregulates NEU3 expression via activating the Janus kinase 2 (JAK2)-STAT5 signalling pathway through its receptor EPOR, thereby to promote the desialylation through enhancing the migration and invasion ability of FLS, which is verified by JAK2 inhibitor and NEU3 inhibitor. CONCLUSION: EPO, as a proinflammatory factor, accelerates the process of RA through transcriptional upregulation of the expression of NEU3 by JAK2/STAT5 pathway.

circTldc1 increases Tldc1 expression by targeting miR-485-5p to promote fibroblast-like synoviocytes proliferation in collagen-induced arthritis.

Abnormalities in the function of fibroblast-like synoviocytes (FLSs) are crucial factors leading to joint damage of rheumatoid arthritis. In recent years, the role of circular RNA (circRNA) in RA has gradually been revealed. However, the functional regulation of FLSs mediated by circRNA and its potential mechanisms remain unclear. In this study, we elucidated the expression profile of circRNA in FLSs, as well as the role and molecular mechanisms of circTldc1. Through sequencing and validation experiments on primary FLSs derived from collagen-induced arthritis (CIA) rats, we found that circTldc1 can promote FLSs proliferation and exacerbate CIA-induced joint damage. The data revealed that circTldc1's parent gene, Tldc1, is homologous to human Tldc1, and circTldc1 is located in the cytoplasm of FLSs, belonging to the exonic circRNA category. The results from bioinformatics analysis, molecular experiments on FLSs (manipulating circTldc1 expression in vitro), and animal experiments (local regulation of circTldc1 expression in vivo) collectively confirmed that circTldc1 promotes Tldc1 expression by targeting miR-485-5p. High expression of Tldc1 further enhances FLSs proliferation and inflammatory responses, thereby worsening joint damage in CIA rats. High expression of circTldc1 and its parent gene Tldc1 may serve as biomarkers for RA. Local regulation of circTldc1 and Tldc1 gene levels in the joint cavity may represent a potential strategy to improve joint damage and inflammation in RA.

FNDC4 reduces inflammation, proliferation, invasion and migration of rheumatoid synovial cells by inhibiting CCL2/ERK signaling.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation. Fibronectin type III domain-containing protein 4 (FNDC4) is a secretory factor that can regulate inflammatory diseases. However, the role of FNDC4 in RA has not been reported so far. METHODS: The expression of FNDC4 in synovial tissues of RA was analyzed by GEO database (GSE55235 dataset). Then, the expression of FNDC4 in RA fibroblast-like synoviocytes (RA-FLSs) was detected by RT-qPCR and western blot. After constructing FNDC4 overexpression plasmid, cell proliferation and apoptosis were detected. Wound healing and transwell assays were used to detect cell migration and invasion. Then we examined the expression of cytokines related to cell inflammation. Subsequently, the regulatory mechanism of FNDC4 was further discussed. We detected the expression of CCL2 and ERK signaling pathway related proteins downstream of FNDC4. Finally, the mechanism was discussed through the overexpression of FNDC4 and CCL2 and the addition of ERK pathway activator tBHQ. RESULTS: GEO database showed that FNDC4 expression decreased in synovial tissues of RA. FNDC4 expression was also decreased in RA-FLSs. Overexpression of FNDC4 inhibited the proliferation, invasion and migration of RA-FLSs whereas promoted the cellapoptosis. Overexpression of FNDC4 inhibited the release of inflammatory factors in RA-FLSs. The regulatory effect of FNDC4 is achieved by inhibiting the CCL2/ERK signaling pathway. CONCLUSION: FNDC4 reduces inflammation, proliferation, invasion and migration of RA-FLSs in RA by inhibiting CCL2/ERK signaling.